Endocrine tumors of the adrenal medulla are known as pheochromocytomas. These tumors are rare in humans, as in most other species, except in patients with hereditary endocrine or neurocutaneous tumor syndromes. Pheochromocytomas are developmentally related to neuroblastomas, which are common adrenal medullary neoplasms, and pheochromocytoma cells can undergo neuronal differentiation. However, an important difference between the endocrine and neuronal phenotype is that the latter is associated with inability to express phenylethanolamine N-methyltransferase (PNMT), the enzyme that synthesizes epinephrine. There are currently no human pheochromocytoma cell lines, and other lines are not adequate models for studying PNMT regulation because they are noradrenergic, and express little or no PNMT. Pheochromocytomas occur with increased frequency in patients with neurofibromatosis type 1, a neurocutaneous syndrome characterized by loss or defective function of neurofibromin, the protein encoded by the tumor suppressor gene NF1. Neurofibromin is a GTPase-activating protein that can regulate the activity of ras proteins in intracellular signalling. However, neurofibromin can inhibit ras-dependent tumor growth independently of its GTPase activity. Transplantable pheochromocytoma tumors and cell lines have now been developed from neurofibromatosis knockout mice. Mouse pheochromocytoma (MPC) cells show spontaneous or growth factor- induced neuronal differentiation in culture similarly to their human counterparts and express PNMT in vivo and in vitro. These tumors have clear relevance to human disease. They are a valuable new tool for studying signalling pathways in adrenal medullary neoplasms, and are a unique model for studying regulation of PNMT expression. The proposed studies will develop and utilize this model with the long-term objective of determining mechanisms responsible for development and progression of adrenal medullary tumors and for regulating tumor cell phenotype. The Specific Aims are: (1) to establish lines of MPC cells characterized with respect to basal expression of PNMT. (2) To establish conditions that promote PNMT expression or neuronal differentiation in MPC cells and test the hypothesis that neuronal differentiation causes loss of PNMT expression. (3) To use MPC cells to test the hypothesis that PNMT expression is determined by an interplay of silencing and positively-acting transcription factors, such that silencing predominates both in noradrenergic pheochromocytomas and in pheochromocytomas that have undergone neuronal differentiation. (4) To determine the status of neurofibromin in MPC cells and test the hypothesis that abnormal regulation of ras activity is responsible for neuronal differentiation in this model.